A preparation method of a reference sample of artemisia soup and a construction method of high performance liquid chromatography characteristic spectrum of the reference sample of artemisia soup

Abstract

The application provides a preparation method of a reference sample of Artemisia capillaris decoction. The application also provides a construction method of a high performance liquid chromatography characteristic map of the reference sample of Artemisia capillaris decoction. The application firstly constructs a characteristic map of Artemisia capillaris decoction by using a high performance liquid chromatography method, and fills the blank of quality control by the method of the characteristic map, realizes comprehensive reflection of the quality condition of the prescription, and makes the quality control more perfect. The method of the application has the advantages of simple operation, good separation degree, high precision, good repeatability and good stability, etc. Under specific detection conditions, the identification of the characteristic map method of Artemisia capillaris decoction can be realized by taking chlorogenic acid as a reference S peak.

Description

Technical Field

[0001] This invention relates to a method for preparing a reference sample of Artemisia capillaris decoction and a method for constructing a high-performance liquid chromatography (HPLC) characteristic spectrum of the reference sample of Artemisia capillaris decoction. Background Technology

[0002] Yin Chen Hao Tang was first recorded in Zhang Zhongjing's *Treatise on Cold Damage*. It consists of three herbs: Artemisia capillaris, Gardenia jasminoides, and Rheum palmatum. The original text states, "Artemisia capillaris 6 liang, Gardenia jasminoides 14 pieces (split), Rheum palmatum 2 liang (peeled). Boil these three herbs in 12 liters of water. First, boil the Artemisia capillaris until the water is reduced by 6 liters. Then add the other two herbs and boil until 3 liters remain. Remove the dregs and divide into three doses." Yin Chen Hao Tang has the effects of clearing heat, promoting diuresis, and relieving jaundice. Its treatment principle combines dampness removal with heat clearing, and promotes diuresis with purgation. It is a basic and representative formula for treating damp-heat jaundice, and also has choleretic, jaundice-relieving, and liver-damaging effects. The principal herb, Artemisia capillaris, has the effect of clearing heat and promoting diuresis. Its main chemical components include flavonoids, coumarins, and organic acids, which have the effects of protecting the liver and gallbladder, anti-inflammation, anti-oxidation, and anti-tumor. The assistant herb, Gardenia jasminoides, can enhance the effect of Artemisia capillaris, promote diuresis and eliminate heat. It contains chemical components such as iridoids, terpenes, organic acid esters, and flavonoids, which have the effects of anti-inflammation, antiviral, and anti-tumor. The adjuvant herb, rhubarb, clears pathogenic heat in the qi level. It contains chemical components such as anthraquinones, anthrones, succinates, and tannins, which have the pharmacological effects of protecting the liver, anti-inflammatory and analgesic, eliminating oxygen free radicals, and anti-tumor. Therefore, the chemical components in Yin Chen Hao Tang can be classified into organic acids, anthraquinones, iridoid glycosides, crocin, and flavonoids. Based on the five principles of Q-marker prediction, chlorogenic acid, cinnamone, crocin I, and emodin methyl ether-8-O-β-D-glucoside contained in Yin Chen Hao Tang can be used as its quality markers, which are closely related to the main efficacy of Yin Chen Hao Tang in "clearing heat, promoting diuresis, and relieving jaundice".

[0003] Liang Hui, et al., Comparison of Chemical Constituents of Yin Chen Hao Tang Prepared by Different Processes, Chinese Patent Medicine, August 2011, Vol. 33, No. 8. This paper examines the differences in chemical composition between combined-decoction granules, separately-decoction granules, and traditional decoction concentrates of Yin Chen Hao Tang, providing a basis for their clinical application from the perspective of chemical composition. Both combined-decoction granules and separately-decoction granules can be used clinically, and the clinical effects of combined-decoction granules may be more consistent with those of traditional decoction concentrates. This literature illustrates the complexity of the components in Yin Chen Hao Tang.

[0004] Application No. 201310139307.5, Invention Title: A Formula Granule of Artemisia capillaris Decoction and its Preparation and Detection Methods. This invention provides a formula granule of Artemisia capillaris decoction, its preparation method, and its detection method. The formula granule is prepared by extracting Artemisia capillaris, Gardenia jasminoides, and Rheum palmatum in a mass ratio of 3:1.5:1 with water, filtering, concentrating, and spray drying. The detection method for the formula granule includes the following steps: identifying the formula granule by thin-layer chromatography; determining the fingerprint spectrum of the formula granule by high-performance liquid chromatography; determining the content of substances in the formula granule by high-performance liquid chromatography; determining the content of leachate in the formula granule by hot immersion method; determining the heavy metal content in the formula granule by residue on ignition test method; and determining the organochlorine pesticide residue content in the formula granule by pesticide residue determination method. The proportions of raw materials in the Yin Chen Hao Tang formula granules in this patent document differ from the original prescription by Zhang Zhongjing. It is intended for the preparation of compound preparations, rather than for a reference sample of Yin Chen Hao Tang.

[0005] Application No. 202210143197.9, Invention Title: A Fingerprint Spectrum Detection Method for Yin Chen Hao Tang Compound Preparation, discloses a fingerprint spectrum detection method for Yin Chen Hao Tang compound preparation. The detection method includes the following steps: S1: Preparation of test solution: Yin Chen, Zhi Zi, and Da Huang are mixed in methanol, ultrasonicated, filtered, concentrated, diluted, and filtered again to obtain the test solution; S2: Preparation of reference solution: The target reference standard is dissolved and filtered to obtain the reference solution; S3: The test solution and reference solution are injected into a liquid chromatograph to obtain chromatograms; the chromatograms are imported into the Chinese Pharmacopoeia Commission's Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System to automatically calculate the similarity; the chromatographic conditions are: C18 column; water as mobile phase A, methanol as mobile phase B; flow rate of 0.8-1.2 mL / min; gradient elution; detection wavelength of 205-280 nm; injection volume of 5-20 μL; column temperature of 25-35℃. The fingerprint spectroscopy detection method described in this invention effectively controls the characteristic components of Yin Chen Hao Tang compound preparations. The fingerprint spectroscopy exhibits high quality, precision, stability, and repeatability. The HPLC detection method established in this patent literature for detecting Yin Chen Hao Tang has an elution time of 105 minutes, which is long and inefficient. Furthermore, the characteristic peaks 1 and 2 of this invention are close in position, and there is no experimental data to indicate whether the resolution is greater than 1.5. Based solely on the attached diagram, the elution time is likely within 5 minutes, and solvent peaks are present.

[0006] In high-performance liquid chromatography (HPLC), the reference sample (standard / reference) is the core of quantitative analysis, and its quality directly affects the accuracy and reliability of the results. Currently, there are no relevant literature reports on the reference sample and quality control methods for Yin Chen Hao Tang (Artemisia capillaris decoction). The preparation of the reference sample and the construction of its characteristic chromatograms for Yin Chen Hao Tang face the following challenges:

[0007] (1) Key information verification: Yin Chen Hao Tang is a classic prescription, selected from ancient Chinese medicine books. The prescription is rigorous. In order to conform to the original prescription of Zhang Zhongjing's "Treatise on Febrile Diseases", key information verification was carried out by combining ancient and modern literature measurement, the historical evolution of Yin Chen Hao Tang, and modern clinical applications.

[0008] (2) Complexity of components: The components of traditional Chinese medicine are complex, and the polarity, molecular weight and chemical properties of various compounds are different, which increases the difficulty of separation and detection.

[0009] (3) Identification and assignment of characteristic peaks: In the determination of characteristic spectra, multiple references are used to identify different characteristic peaks. In addition to evaluating the retention time, relative retention time / relative peak area, etc., the identified characteristic peaks also need to be confirmed by spectral analysis. For traditional Chinese medicine compound preparations, the characteristic peaks also need to be assigned to the medicinal ingredients based on the chemical components and preparation process of each ingredient in the prescription. Summary of the Invention

[0010] This invention relates to a method for preparing a reference sample of Artemisia capillaris decoction and a method for constructing a high-performance liquid chromatography (HPLC) characteristic spectrum of the reference sample of Artemisia capillaris decoction.

[0011] This invention provides a method for preparing a reference sample of Artemisia capillaris decoction, which includes the following steps:

[0012] a. Weigh the raw materials according to the following weight ratios: Artemisia capillaris 82.8g, Gardenia jasminoides 12.0g, Rheum palmatum 27.6g;

[0013] b. Add water, first boil Artemisia capillaris, then add Gardenia jasminoides and Rheum palmatum, decoct, filter, collect the liquid, freeze dry using a vacuum freeze dryer to obtain freeze-dried powder;

[0014] The freeze-drying process of the vacuum freeze dryer is as follows:

[0015] (1) Pre-freezing: Temperature: -50℃, holding temperature: 3h; Pressure: atmospheric pressure;

[0016] (2) Sublimation drying: Temperature: -40℃, holding temperature: 1h; Pressure: 0.0pa;

[0017] Temperature: -30℃, temperature maintenance: 1h; Pressure: 0.0pa;

[0018] Temperature: -20℃, temperature maintenance: 2h; Pressure: 0.0pa;

[0019] Temperature: -10℃, temperature maintenance: 3h; Pressure: 0.0pa;

[0020] Temperature: 0℃, temperature maintenance: 3h; Pressure: 0.0pa;

[0021] (3) Desorption drying: Temperature: 10℃, holding temperature: 3h; Pressure: 0.0pa;

[0022] Temperature: 20℃, temperature maintenance: 2h; Pressure: 0.0pa;

[0023] Temperature: 30℃, temperature maintenance: 2h; Pressure: 0.0pa;

[0024] Temperature: 40℃, temperature maintenance: 2h; pressure: 0.0pa.

[0025] Preferably, in step b, add 20 times the amount of water, first boil Artemisia capillaris until the water volume is 10 times, then add Gardenia jasminoides and Rheum palmatum, boil until the water volume is 5 times, filter through a 200-mesh sieve, and collect the medicinal liquid.

[0026] The present invention also provides a method for preparing the Artemisia capillaris decoction reference sample to obtain the Artemisia capillaris decoction reference sample.

[0027] This invention also provides a method for constructing the high-performance liquid chromatography (HPLC) characteristic spectrum of the Artemisia capillaris decoction reference sample, which includes the following steps:

[0028] a. Preparation of the test solution:

[0029] The reference sample of Artemisia capillaris decoction was dissolved and extracted with a solvent to obtain the test solution;

[0030] b. The test solution was analyzed by high-performance liquid chromatography (HPLC) to obtain the HPLC characteristic chromatogram of the Artemisia capillaris decoction reference sample; the HPLC chromatographic conditions were as follows: C18 column; mobile phase A was acetonitrile, mobile phase B was 0.1% phosphoric acid, and gradient elution was performed; the gradient elution conditions were as follows:

[0031] From 0 to 6 minutes, mobile phase A is 2% to 10%, and mobile phase B is 98% to 90%.

[0032] 6–13 min, mobile phase A is 10–15%, mobile phase B is 90–85%.

[0033] 13–60 min, mobile phase A is 15–35%, mobile phase B is 85–65%.

[0034] The preparation of the test solution is as follows: take the freeze-dried powder of Artemisia capillaris decoction, add methanol aqueous solution, sonicate, cool to room temperature, filter, and take the filtrate to obtain the test solution.

[0035] It also includes the preparation of reference solutions: chlorogenic acid, neochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, gallic acid, catechin and rhein-8-O-β-D-glucoside reference standards are dissolved in methanol to prepare reference solutions.

[0036] The chromatographic column is a C18 column with a length of 250 mm, an inner diameter of 4.6 mm, and a silica gel particle size of 5 μm; the detector is a diode array detector with a detection wavelength of 285 nm; the column temperature is 30 °C; and the flow rate is 1.0 mL / min.

[0037] The chromatogram of the test sample contains 14 characteristic peaks, among which the peak corresponding to the chlorogenic acid reference is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within ±10% of the specified value. The specified values ​​of each peak are as follows: Peak 1 0.498, Peak 2 0.709, Peak 3 0.735, Peak 4 0.974, Peak 5 (S peak) 1.000, Peak 6 1.112, Peak 7 1.313, Peak 8 1.653, Peak 9 1.965, Peak 10 2.100, Peak 11 2.237, Peak 12 2.565, Peak 13 2.614, Peak 14 3.050.

[0038] Among them, peak 1 is gallic acid, peak 3 is neochlorogenic acid, peak 4 is catechin, peak 5 is chlorogenic acid, peak 10 is isochlorogenic acid B, peak 11 isochlorogenic acid A, peak 12 isochlorogenic acid C, and peak 14 is emodin-8-O-β-D-glucoside.

[0039] This invention also provides a method for judging the quality of Artemisia capillaris decoction, which includes the following steps:

[0040] a. Take Artemisia capillaris decoction as the test sample;

[0041] b. The method for constructing the high-performance liquid chromatography characteristic spectrum of the Yin Chen Hao Tang reference sample is used to test the sample. If the sample contains 14 characteristic peaks, it is a qualified Yin Chen Hao Tang sample.

[0042] The present invention establishes a preparation method for a reference sample of Yinchenhao Decoction. According to the original records, key information is verified by combining ancient and modern bibliometrics. One liang in the Eastern Han Dynasty is equivalent to 13.8 g in modern times, and one sheng of water is equivalent to 200 ml in modern times. That is, the decoction method of the reference sample of Yinchenhao Decoction is: "Take 82.8 g of Artemisiae Scopariae Herba, 12.0 g of Gardeniae Fructus, and 27.6 g of Rhei Radix et Rhizoma, add 2400 ml of water, decoct Artemisiae Scopariae Herba first until the volume reaches 1200 ml, then add Gardeniae Fructus and Rhei Radix et Rhizoma, continue decocting until the volume reaches 600 ml, filter, and collect the medicinal liquid to obtain the reference sample." The mass ratio of Artemisiae Scopariae Herba, Gardeniae Fructus and Rhei Radix et Rhizoma is 6.9:1:2.3, and the mixture is decocted with 20 times the amount of water. The proportion of herbal medicines in the disclosed patent documents is different from the original intention of Zhang Zhongjing's original prescription, which aims at the preparation of compound preparations rather than the reference sample of Yinchenhao Decoction.

[0043] At present, there are few studies on the characteristic chromatogram or fingerprint of Yinchenhao Decoction. The present invention simultaneously adopts high performance liquid chromatography to establish a characteristic chromatogram detection method for the reference sample of Yinchenhao Decoction. Among the identified components, organic acid components account for 62.5%, and organic acids are related to the effects of clearing heat, promoting diuresis and benefiting gallbladder of Artemisiae Scopariae Herba, and have obvious effects such as antivirus, liver protection and choleresis. In addition, the identification of p-hydroxyanthraquinones and polyphenols enables the identification of key components of Yinchenhao Decoction. In addition, Yinchenhao Decoction has complex components and contains many water-soluble components. When establishing the detection method, the initial proportion of the water phase in gradient elution is set to 98%, which can better separate components with high polarity, has good system suitability, is a simpler and more efficient detection method, and is of great significance for evaluating the reference sample of Yinchenhao Decoction.

[0044] The beneficial effects of the technical solution are as follows:

[0045] (1) The present invention uses high performance liquid chromatography to construct the characteristic chromatogram of Yinchenhao Decoction for the first time, and through this characteristic chromatogram method, fills the gap in its quality control, can comprehensively reflect the quality status of the prescription comprehensively, improves the perfection of its quality control, and adds a new means for its quality standard.

[0046] (2) The method of the present invention adopts gradient elution for determination by high performance liquid chromatography, which has short detection time and is more efficient and rapid; it has the advantages of simple operation, good resolution, high precision, good repeatability and good stability. Under specific detection conditions, by taking the chlorogenic acid chromatographic peak as the reference peak S, the identification of Yinchenhao Decoction by the characteristic chromatogram method can be realized. Description of Drawings

[0047] Figure 1 are characteristic chromatograms of Yinchenhao Decoction under investigation of different flow rates;

[0048] Figure 2 are characteristic chromatograms of Yinchenhao Decoction under investigation of different column temperatures;

[0049] Figure 3Characteristic chromatograms of Yin Chen Hao Tang under different injection volumes were obtained.

[0050] Figure 4 Characteristic chromatograms for different extraction methods of Yin Chen Hao Tang;

[0051] Figure 5 Characteristic spectra of Yin Chen Hao Tang under different solvents;

[0052] Figure 6 Characteristic chromatograms for investigating different solvent dosages in Yin Chen Hao Tang;

[0053] Figure 7 Characteristic chromatograms for different extraction times of Yin Chen Hao Tang;

[0054] Figure 8 Chromatographic peak identification diagram during the detection of Yin Chen Hao Tang;

[0055] Figure 9 A negative control identification diagram used in the testing of Yin Chen Hao Tang (Artemisia capillaris decoction);

[0056] Figure 10 Characteristic spectrum of the delay when detecting Yin Chen Hao Tang;

[0057] Figure 11 Characteristic spectra of Yin Chen Hao Tang obtained by different instruments;

[0058] Figure 12 Characteristic chromatograms of Yin Chen Hao Tang were obtained by examining different chromatographic columns.

[0059] Figure 13 A characteristic chromatogram of Yin Chen Hao Tang (Artemisia capillaris decoction) as a control. Detailed Implementation

[0060] The experimental instruments and materials involved in the following examples are as follows (all percentages are based on mass concentration):

[0061] High-performance liquid chromatographs: Agilent 1260 high-performance liquid chromatograph, Shimadzu LC-20AD high-performance liquid chromatograph, Waters e2695 high-performance liquid chromatograph;

[0062] Electronic balances: ME204E / 02, XP26 (Mettler-Toledo Instruments Ltd.);

[0063] Ultrapure water system: Cellular type 1810A (Shanghai Moler Scientific Instruments Co., Ltd.);

[0064] Ultrasonic cleaner: KQ5200DB model (600W, 40kHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0065] Column: Agilent ZORBAX SB-Aq 250×4.6mm, 5µm, Welch AQ-C18 250×4.6mm,5um, Ecosil 120-5-AQ PLUS250×4.6mm,5um;

[0066] Acetonitrile and phosphoric acid were of chromatographic grade, water was ultrapure water, and all other reagents were of analytical grade.

[0067] Chlorogenic acid (China National Institutes for Food and Drug Control, batch number: 110753-202119, content calculated as 96.3%);

[0068] New chlorogenic acid (Chengdu Desite Biotechnology Co., Ltd., batch number: MUST-21030108, content calculated as 99.67%);

[0069] Isochlorogenic acid A (Chengdu Desite Biotechnology Co., Ltd., batch number: MUST-18032601, content calculated as 98.82%);

[0070] Isochlorogenic acid B (Chengdu Desite Biotechnology Co., Ltd., batch number: DST210823-037, content calculated as 98%);

[0071] Isochlorogenic acid C (Chengdu Desite Biotechnology Co., Ltd., batch number: DSTDY003804, content calculated as 98%);

[0072] Gallic acid (China National Institutes for Food and Drug Control, batch number: 110831-202407, content calculated as 91.5%);

[0073] Catechins (China National Institutes for Food and Drug Control, batch number: 110877-202306, content calculated as 96.6%);

[0074] Emodin-8-O-β-D-glucoside (Sichuan Weikeqi Biotechnology Co., Ltd., batch number: wkq18082803, content is 98%);

[0075] Artemisia capillaris (batch number: 010720-2307001);

[0076] Gardenia (batch number: 010533-1901003);

[0077] Rhubarb (batch number: 010097-2110001);

[0078] Artemisia capillaris decoction (batch number: YCHT-JZYP-01).

[0079] Example 1

[0080] This embodiment relates to Yin Chen Hao Tang (Artemisia capillaris decoction).

[0081] (1) The preparation of Yin Chen Hao Tang freeze-dried powder is as follows: 82.8g of Yin Chen, 12.0g of Gardenia, 27.6g of Rhubarb, add 20 times the amount of water, first boil Yin Chen until the amount of water is 10 times, then add Gardenia and Rhubarb, boil until the amount of water is 5 times, filter with a 200-mesh sieve, collect the medicinal liquid, freeze dry with a vacuum freeze dryer to obtain freeze-dried powder.

[0082] The freeze-drying process of the vacuum freeze dryer is as follows:

[0083] (1) Pre-freezing: Temperature: -50℃, holding temperature: 3h; Pressure: atmospheric pressure;

[0084] (2) Sublimation drying: Temperature: -40℃, holding temperature: 1h; Pressure: 0.0pa;

[0085] Temperature: -30℃, temperature maintenance: 1h; Pressure: 0.0pa;

[0086] Temperature: -20℃, temperature maintenance: 2h; Pressure: 0.0pa;

[0087] Temperature: -10℃, temperature maintenance: 3h; Pressure: 0.0pa;

[0088] Temperature: 0℃, temperature maintenance: 3h; Pressure: 0.0pa;

[0089] (3) Desorption drying: Temperature: 10℃, holding temperature: 3h; Pressure: 0.0pa;

[0090] Temperature: 20℃, temperature maintenance: 2h; Pressure: 0.0pa;

[0091] Temperature: 30℃, temperature maintenance: 2h; Pressure: 0.0pa;

[0092] Temperature: 40℃, holding temperature: 2h; Pressure: 0.0pa. Freeze-drying technology can better preserve the original form of materials and their nutrients, obtaining high-quality dried products. Reference samples are a key carrier bridging traditional Chinese medicine preparations and ancient classic prescriptions. The choice of their material form directly affects the key quality attributes of the reference samples and preparations, and is also the key to "reproducing a bowl of soup" in the study of classic prescriptions. Therefore, freeze-drying is generally used to prepare reference samples to maximize the preservation of the effective components and yield of classic prescriptions.

[0093] The preparation method of the test solution includes: taking 0.2g of Artemisia capillaris decoction freeze-dried powder, adding 25mL of 70% methanol, sonicating (power 600W, frequency 40kHz), cooling to room temperature, filtering with a 0.45μm filter screen, and collecting the filtrate to obtain the test solution.

[0094] (2) Preparation of reference solution: Take chlorogenic acid, neochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, gallic acid, catechin and rhein-8-O-β-D-glucoside reference standards, dissolve them in methanol to prepare a mixed reference solution with a content of 100ug per 1mL of methanol solution.

[0095] (3) The test solution obtained in step (1) and the reference solution obtained in step (2) are subjected to high performance liquid chromatography to obtain the corresponding characteristic chromatograms.

[0096] The high-performance liquid chromatography (HPLC) detection conditions are met:

[0097] Liquid chromatography column: C18 column;

[0098] Column temperature: 30℃;

[0099] Flow rate: 1.0 mL / min;

[0100] Detection wavelength: 285nm;

[0101] Mobile phase A is acetonitrile, and mobile phase B is 0.1% phosphoric acid, with gradient elution.

[0102] The gradient rinsing process is as follows:

[0103] From 0 to 6 minutes, mobile phase A is 2% to 10%, and mobile phase B is 98% to 90%.

[0104] 6–13 min, mobile phase A is 10–15%, mobile phase B is 90–85%.

[0105] 13–60 min, mobile phase A is 15–35%, mobile phase B is 85–65%.

[0106] (4) Using the characteristic spectrum of the reference solution in step (3) as a reference spectrum, select common peaks from the characteristic spectrum of the test solution to construct the characteristic spectrum of Yin Chen Hao Tang.

[0107] The constructed characteristic spectrum of Yin Chen Hao Tang (Artemisia capillaris decoction) has 14 characteristic peaks, among which the peak corresponding to the chlorogenic acid reference is the S peak. The relative retention time of each characteristic peak and the S peak was calculated, and the relative retention time should be within ±10% of the specified value. Peak 1 0.498, peak 2 0.709, peak 3 0.735, peak 4 0.974, peak 5 (S peak) 1.000, peak 6 1.112, peak 7 1.313, peak 8 1.653, peak 9 1.965, peak 10 2.100, peak 11 2.237, peak 12 2.565, peak 13 2.614, peak 14 3.050.

[0108] Example 2

[0109] System applicability assessment

[0110] (1) Investigation of different flow velocities

[0111] Using the test solution of Yin Chen Hao Tang described in Example 1 as the object, different flow rates of 0.8 ml / min, 1.0 ml / min, and 1.2 ml / min were compared. The results showed that the chromatographic baseline was more stable at a flow rate of 1.0 ml / min; therefore, a flow rate of 1.0 ml / min was selected. Figure 1 As shown.

[0112] (2) Investigation at different column temperatures

[0113] Using the test solution of Yin Chen Hao Tang described in Example 1 as the object, comparisons were made at different column temperatures of 25℃, 30℃, and 35℃. The results showed that the chromatogram baseline was more stable at a column temperature of 30℃. Therefore, the flow rate was set at a column temperature of 30℃. Figure 2 As shown.

[0114] (3) Investigation of different injection volumes

[0115] Using the test solution of Yin Chen Hao Tang described in Example 1 as the object, different injection volumes of 5 μL, 10 μL, and 20 μL were compared. The results showed that the chromatographic baseline was more stable when the injection volume was 10 μL; therefore, the injection volume was 10 μL. Figure 3 As shown.

[0116] Example 3

[0117] Examination of different extraction methods

[0118] Using the sample solution of Yin Chen Hao Tang described in Example 1 as the object, different extraction methods, such as reflux treatment and ultrasonic treatment, were compared. The results showed that the chromatograms of the two extraction methods had high similarity. Since ultrasonic treatment is simpler to operate, it was chosen as the extraction method. Figure 4 As shown.

[0119] Example 4

[0120] Investigation of different extraction solvents

[0121] Using the Artemisia capillaris decoction sample solution described in Example 1 as the test sample, 25 mL of water, 30% methanol, 70% methanol, and methanol (different extraction solvents) were added respectively. The mixture was ultrasonically treated (600 W power, 40 kHz frequency) for 30 minutes, cooled to room temperature, and filtered through a 0.45 μm filter. The filtrate was collected. The results showed that each chromatographic peak was extracted better in 70% methanol; therefore, 70% methanol was selected as the extraction solvent. Figure 5 As shown.

[0122] Example 5

[0123] Investigation of different solvent dosages

[0124] Using the Artemisia capillaris decoction sample solution described in Example 1 as the test sample, different solvent volumes of 10 mL, 25 mL, and 50 mL of 70% methanol were added, respectively. The mixture was ultrasonically treated (power 600 W, frequency 40 kHz) for 30 minutes, cooled to room temperature, and filtered through a 0.45 μm filter. The filtrate was collected. The results showed that the separation of each chromatographic peak was better when 25 mL of 70% methanol was added for extraction. Therefore, 25 mL of solvent was selected. Figure 6 As shown.

[0125] Example 6

[0126] Examination of different extraction times

[0127] Using the Artemisia capillaris decoction sample solution described in Example 1 as the test sample, 25 mL of 70% methanol was added, and the mixture was ultrasonically treated (600 W power, 40 kHz frequency) for 20 minutes, 30 minutes, and 40 minutes respectively. After cooling to room temperature, the solution was filtered through a 0.45 μm filter, and the filtrate was collected. The results showed that the extraction time had little effect on the chromatographic peaks; therefore, an extraction time of 30 minutes was selected. Figure 7 As shown.

[0128] Example 7

[0129] Identification of chromatographic peaks

[0130] Using the test solution of Artemisia capillaris decoction described in Example 1 as the object, different reference standards (chlorogenic acid reference solution, neochlorogenic acid reference solution, isochlorogenic acid A reference solution, isochlorogenic acid B reference solution, isochlorogenic acid C reference solution, gallic acid reference solution, catechin reference solution, and emodin-8-O-β-D-glucoside reference solution) were compared. Peak 1 was identified as gallic acid, peak 3 as neochlorogenic acid, peak 4 as catechin, peak 5 as chlorogenic acid, peak 10 as isochlorogenic acid B, peak 11 as isochlorogenic acid A, peak 12 as isochlorogenic acid C, and peak 14 as emodin-8-O-β-D-glucoside. Figure 8 As shown.

[0131] Example 8

[0132] Negative control identification

[0133] Using the test solution of Yin Chen Hao Tang described in Example 1 as the object, and employing the same preparation method, negative control sample solutions (Yin Chen negative control, Gardenia negative control, and Rhubarb negative control) lacking one of the herbs were prepared for comparison. It was found that the chromatographic peaks exhibited by this method mainly originated from the principal herb, Yin Chen, further demonstrating the significance of this method for the quality evaluation of Yin Chen Hao Tang. Figure 9 As shown.

[0134] Example 9

[0135] Precision examination

[0136] Accurately weigh 0.2g of Artemisia capillaris decoction freeze-dried powder, prepare the test solution according to the method described in Example 1, and inject it into the liquid chromatograph. The injection was repeated 6 times. The relative retention time (RSD%) of each characteristic peak was calculated to be <2%, indicating that the instrument has good precision.

[0137] Table 1 Precision test – Relative retention time

[0138]

[0139]

[0140] Example 10

[0141] Repeated examination

[0142] Accurately weigh 0.2g of each of 6 portions of Artemisia capillaris decoction freeze-dried powder, prepare the test solution according to the method described in Example 1, and inject it into the liquid chromatograph. The relative retention time (RSD%) of each characteristic peak is calculated to be <5%, indicating that the method has good repeatability.

[0143] Table 2. Repeatability Tests – Relative Retention Time

[0144]

[0145] Example 11

[0146] Stability assessment

[0147] Accurately weigh 0.2 g of Artemisia capillaris decoction freeze-dried powder and prepare the test solution according to the method described in Example 1. The test solution was measured at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h. The relative retention time (RSD%) of each characteristic peak was calculated to be <5%, indicating that the test solution was stable within 24 hours.

[0148] Table 3 Stability Study – Relative Retention Time

[0149]

[0150] Example 12

[0151] Delayed examination

[0152] Using the test solution of Yin Chen Hao Tang described in Example 1 as the object, based on the original elution gradient, isocratic elution with mobile phase A at 35% and mobile phase B at 65% was added for 60-120 min to investigate the elution delay. The results showed that no chromatographic peak eluted after a 60-minute delay, indicating that the original gradient had a good elution effect on Yin Chen Hao Tang. Figure 10 As shown.

[0153] Example 13

[0154] Durability assessment

[0155] (1) Investigations by different personnel and at different times

[0156] Two samples of 0.2 g of Artemisia capillaris decoction freeze-dried powder were accurately weighed by different personnel (A and B) at different times (T1 and T2) to prepare test samples for determination. The relative retention time (RSD%) of each characteristic peak was calculated to be <5%, indicating that the method has good stability.

[0157] Table 4. Personnel and Time Assessment – ​​Relative Retention Time

[0158]

[0159] (2) Investigation with different instruments

[0160] Using the Artemisia capillaris decoction test solution described in Example 1 as the object, comparisons were made using an Agilent 1260 high-performance liquid chromatograph, a Shimadzu LC-20AD high-performance liquid chromatograph, and a Waters e2695 high-performance liquid chromatograph, respectively. Figure 11 As shown.

[0161] (3) Investigation of different chromatographic columns

[0162] Using the test solution of Yin Chen Hao Tang described in Example 1 as the object, different chromatographic columns with a column length of 250 mm, an inner diameter of 4.6 mm, and a silica gel particle size of 5 μm were used: Agilent ZORBAX SB-Aq and Welch. A comparison was made between AQ-C18 and Ecosil 120-5-AQ PLUS, such as... Figure 12 As shown.

[0163] Example 14

[0164] Verification and Investigation of Yin Chen Hao Tang

[0165] The characteristic chromatographic method was investigated for Yin Chen Hao Tang and mixed reference solutions, and the corresponding characteristic spectra were obtained, such as... Figure 13 As shown.

[0166] From the above Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8 and Figure 9It is known that this invention establishes a method for analyzing the characteristic chromatogram of Yin Chen Hao Tang by analyzing HPLC chromatograms. A total of 14 characteristic peaks were detected in the chromatogram of Yin Chen Hao Tang. This method establishes a high-performance liquid chromatography characteristic chromatogram method for Yin Chen Hao Tang. This method is used to evaluate Yin Chen Hao Tang. At the same time, the retention time of the 14 characteristic peaks detected by this method is within 60 minutes, and the methodological results are good. This reflects the gap in the quality evaluation of this prescription and adds another means to its quality control.

[0167] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing a high-performance liquid chromatography (HPLC) characteristic spectrum of a Artemisia capillaris decoction reference sample, characterized in that: It includes the following steps: a. Preparation of the test solution: The preparation of the test solution is as follows: take the freeze-dried powder of Artemisia capillaris decoction, add methanol aqueous solution, sonicate, cool to room temperature, filter, and take the filtrate to obtain the test solution. b. The test solution was analyzed by high-performance liquid chromatography (HPLC) to obtain the HPLC characteristic chromatogram of the Artemisia capillaris decoction reference sample; the HPLC chromatographic conditions were as follows: C18 column; mobile phase A was acetonitrile, mobile phase B was 0.1% phosphoric acid, and gradient elution was performed; the gradient elution conditions were as follows: From 0 to 6 minutes, mobile phase A is 2% to 10%, and mobile phase B is 98% to 90%. 6–13 min, mobile phase A is 10–15%, mobile phase B is 90–85%. 13–60 min, mobile phase A is 15–35%, mobile phase B is 85–65%; The preparation method of the Artemisia capillaris decoction reference sample includes the following steps: a. Weigh the raw materials according to the following weight ratios: Artemisia capillaris 82.8g, Gardenia jasminoides 12.0g, Rheum palmatum 27.6g; b. Add water, first boil Artemisia capillaris, then add Gardenia jasminoides and Rheum palmatum, decoct, filter, collect the liquid, freeze dry using a vacuum freeze dryer to obtain freeze-dried powder; The freeze-drying process of the vacuum freeze dryer is as follows: (1) Pre-freezing: Temperature: -50℃, holding temperature: 3h; Pressure: atmospheric pressure; (2) Sublimation drying: Temperature: -40℃, holding temperature: 1h; Pressure: 0.0pa; Temperature: -30℃, temperature maintenance: 1h; Pressure: 0.0pa; Temperature: -20℃, temperature maintenance: 2h; Pressure: 0.0pa; Temperature: -10℃, temperature maintenance: 3h; Pressure: 0.0pa; Temperature: 0℃, temperature maintenance: 3h; Pressure: 0.0pa; (3) Desorption drying: Temperature: 10℃, holding temperature: 3h; Pressure: 0.0pa; Temperature: 20℃, temperature maintenance: 2h; Pressure: 0.0pa; Temperature: 30℃, temperature maintenance: 2h; Pressure: 0.0pa; Temperature: 40℃, temperature maintenance: 2h; Pressure: 0.0pa; It also includes the preparation of reference solutions: taking chlorogenic acid, neochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, gallic acid, catechin and rhein-8-O-β-D-glucoside reference standards, dissolving them in methanol to prepare reference solutions; The chromatogram of the test sample contains 14 characteristic peaks, among which the peak corresponding to the chlorogenic acid reference is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within ±10% of the specified value. The specified values ​​of each peak are as follows: Peak 1 0.498, Peak 2 0.709, Peak 3 0.735, Peak 4 0.974, Peak 5 (S peak) 1.000, Peak 6 1.112, Peak 7 1.313, Peak 8 1.653, Peak 9 1.965, Peak 10 2.100, Peak 11 2.237, Peak 12 2.565, Peak 13 2.614, Peak 14 3.

050.

2. The method for constructing the high-performance liquid chromatography characteristic spectrum of the Artemisia capillaris decoction reference sample according to claim 1, characterized in that: In the preparation method of the Artemisia capillaris decoction reference sample, step b involves adding 20 times the amount of water, first boiling Artemisia capillaris until the water volume is 10 times, then adding Gardenia jasminoides and Rheum palmatum, boiling until the water volume is 5 times, filtering through a 200-mesh sieve, and collecting the decoction.

3. The method for constructing the high-performance liquid chromatography characteristic spectrum of the Artemisia capillaris decoction reference sample according to claim 1, characterized in that: The chromatographic column is a C18 column with a length of 250 mm, an inner diameter of 4.6 mm, and a silica gel particle size of 5 μm; the detector is a diode array detector with a detection wavelength of 285 nm; the column temperature is 30 °C; and the flow rate is 1.0 mL / min.

4. The method for constructing the high-performance liquid chromatography characteristic spectrum of the Artemisia capillaris decoction reference sample according to claim 1, characterized in that: Peak 1 is gallic acid, peak 3 is neochlorogenic acid, peak 4 is catechin, peak 5 is chlorogenic acid, peak 10 is isochlorogenic acid B, peak 11 isochlorogenic acid A, peak 12 isochlorogenic acid C, and peak 14 is emodin-8-O-β-D-glucoside.

5. A method for judging the quality of Artemisia capillaris decoction, characterized in that: It includes the following steps: a. Take Artemisia capillaris decoction as the test sample; b. The test sample is tested using the high-performance liquid chromatography characteristic spectrum of the reference sample of Yin Chen Hao Tang according to any one of claims 1-4. If the sample contains 14 characteristic peaks, it is a qualified product of Yin Chen Hao Tang.

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